Droplet-Based Microfluidic Preparation of Shape-Variable Alginate Hydrogel Magnetic Micromotors

This article introduces a facile droplet-based microfluidic method for the preparation of Fe3O4-incorporated alginate hydrogel magnetic micromotors with variable shapes. By using droplet-based microfluidics and water diffusion, monodisperse (quasi-)spherical microparticles of sodium alginate and Fe3O4 (Na-Alg/Fe3O4) are obtained. The diameter varies from 31.9 to 102.7 µm with the initial concentration of Na-Alginate in dispersed fluid ranging from 0.09 to 9 mg/mL. Calcium chloride (CaCl2) is used for gelation, immediately transforming Na-Alg/Fe3O4 microparticles into Ca-Alginate hydrogel microparticles incorporating Fe3O4 nanoparticles, i.e., Ca-Alg/Fe3O4 micromotors. Spherical, droplet-like, and worm-like shapes are yielded depending on the concentration of CaCl2, which is explained by crosslinking and anisotropic swelling during the gelation. The locomotion of Ca-Alg/Fe3O4 micromotors is activated by applying external magnetic fields. Under the rotating magnetic field (5 mT, 1–15 Hz), spherical Ca-Alg/Fe3O4 micromotors exhibit an average advancing velocity up to 158.2 ± 8.6 µm/s, whereas worm-like Ca-Alg/Fe3O4 micromotors could be rotated for potential advancing. Under the magnetic field gradient (3 T/m), droplet-like Ca-Alg/Fe3O4 micromotors are pulled forward with the average velocity of 70.7 ± 2.8 µm/s. This article provides an inspiring and timesaving approach for the preparation of shape-variable hydrogel micromotors without using complex patterns or sophisticated facilities, which holds potential for biomedical applications such as targeted drug delivery.


Introduction
Micromotors are microdevices capable of converting the energy from the environment into autonomous motion. Micromotors show promising aspects for applications in biomedical engineering and environmental remediation [1][2][3][4][5]. Magnetic micromotors are propelled by external magnetic fields. Compared with other types of micromotors, magnetic micromotors have advantages such as remote control, fuel-free, recyclability, etc. [6]. In addition, magnetic micromotors are demonstrated to be excellent candidates for long-term navigation [7]. Various methods have been reported for the preparation of magnetic micromotors with different shapes. Spherical magnetic micromotors can be prepared via the template-assisted method with sputtering or incorporating magnetic materials [8][9][10]. Helical magnetic micromotors can be prepared by two-photon polymerization lithography [11] and 3D printing [12]. Other shapes are produced by using a specific pattern [10,13]. However, these methods require sophisticated facilities and are usually time-consuming. Therefore, they are difficult to be replicated in normal laboratories. On the other hand, these micromotors are commonly composed of rigid materials which are not biodegradable in aqueous environments [14,15]. Thus, facile fabrication methods are in demand for magnetic micromotors using soft biodegradable materials.
Droplet-based microfluidics is widely used for the preparation of monodisperse microparticles. Recently, it attracted much researchers' attention for the fabrication of magnetic further observation ( Figure S1) and removal. With the pressure of nitrogen (50 mbar), the solution of Na-Alg/Fe3O4 and DMC were, respectively, injected into the microchip through two inlets. The flowrate of each fluid was controlled by a gas pump (ELVEFLOW). Due to the immiscibility between these two fluids, at the T-junction on chip (position 1 on the microchip in Figure 1), droplets of Na-Alg/Fe3O4 were generated in DMC, followed by droplet shrinkage. The principle of droplet shrinkage has been reported in our previous article [23]. Briefly, despite the immiscibility between DMC and water, DMC has a low solubility of water (3 wt%) [24]. Thus, after the droplet generation, water diffused gradually from each droplet to DMC, causing droplet shrinkage. It was observed by comparing the droplet at the T-junction (position 1 on chip in Figure 1) and that near the outlet of microchip (position 2 on chip in Figure 1). This process is called onchip droplet shrinkage to indicate that it happens in the microchip. Droplets were collected with the yield of 1 droplet/s in a glass Petri dish filled with DMC through a capillary tube (length 46 cm). Note that the yield varies with experimental parameters, such as the gas pressure, the microchannel dimension, etc. Droplet shrinkage continued out of the microchip (off-chip droplet shrinkage) until the equilibrium was reached. Finally, Na-Alg/Fe3O4 microparticles were obtained. Note that the serpentine channel in the microchip was specially designed to prolong the residence time of droplet ( ). With the straight channel, was relatively short. On-chip droplet shrinkage was not efficient. Large droplets were observed at the outlet of microchip. Besides, they were likely to stagnate there where a capillary tube was connected, probably due to the imperfect connection or a minor flaw in the capillary tube. With water diffusion, the stagnating droplets became condensed and at last clogged the channel. By using the serpentine channel, was prolonged. It allowed for an efficient on-chip droplet shrinkage. Droplets were smaller at the outlet of microchip. Therefore, they were less likely to stagnate or eventually cause clogging there. Moreover, with an elevated droplet shrinkage, the distance between two droplets was also prolonged. At the end of the capillary tube in the glass Petri dish, there was less tendency for droplet coalescence, which was advantageous for preparing monodisperse microparticles.

Preparation of Ca-Alg/Fe3O4 Micromotors via Ionic Crosslinking
Na-Alg/Fe3O4 microparticles were dried in air with the evaporation of DMC. An aqueous solution of CaCl2 (10, 1, 0.1, 0.01 wt%) was added, transforming Na-Alginate into Note that the serpentine channel in the microchip was specially designed to prolong the residence time of droplet (τ droplet ). With the straight channel, τ droplet was relatively short. On-chip droplet shrinkage was not efficient. Large droplets were observed at the outlet of microchip. Besides, they were likely to stagnate there where a capillary tube was connected, probably due to the imperfect connection or a minor flaw in the capillary tube. With water diffusion, the stagnating droplets became condensed and at last clogged the channel. By using the serpentine channel, τ droplet was prolonged. It allowed for an efficient on-chip droplet shrinkage. Droplets were smaller at the outlet of microchip. Therefore, they were less likely to stagnate or eventually cause clogging there. Moreover, with an elevated droplet shrinkage, the distance between two droplets was also prolonged. At the end of the capillary tube in the glass Petri dish, there was less tendency for droplet coalescence, which was advantageous for preparing monodisperse microparticles.

Preparation of Ca-Alg/Fe 3 O 4 Micromotors via Ionic Crosslinking
Na-Alg/Fe 3 O 4 microparticles were dried in air with the evaporation of DMC. An aqueous solution of CaCl 2 (10, 1, 0.1, 0.01 wt%) was added, transforming Na-Alginate into Ca-Alginate hydrogel via ionic crosslinking. Fe 3 O 4 nanoparticles were incorporated inside. The ensemble was used as the hydrogel magnetic micromotor and named for short as Ca-Alg/Fe 3 O 4 micromotor thereafter. It should be mentioned that the gelation process is immediate. Thus, the production of Ca-Alg/Fe 3 O 4 micromotors mainly depends on the microfluidic experimental parameters and the quantity of micromotors required. Scanning electron microscope (SEM, Phenom ProX, Phenom-World, The Netherlands) was used to characterize the morphology of Na-Alg/Fe 3 O 4 microparticles and Ca-Alg/Fe 3 O 4 micromotors. The SEM sample was prepared by adhering dried particles to a copper sample-holder with a conductive tape. Some particles were broken after this step, which revealed information for the interior structure [23]. For a better resolution, all samples were coated by a thin layer of gold (Au) before SEM observation. The acceleration voltage of SEM was 15 kV. Energy-dispersive X-ray (EDX, Phenom) mapping analysis was conducted for element detection.

Locomotion of Ca-Alg/Fe 3 O 4 Micromotors in External Magnetic Fields
A home-made magnetic field generator was constructed [25]. It consisted of electromagnetic coils, a function generator (FY8300S) and a power amplifier (HSLFSun GLY-FP1000, Lab.Gruppen, Kungsbacka, Sweden). It allowed control of the magnetic field strength and orientation in the X, Y and Z axes by modulating the current in each electromagnetic coil. Sinusoidal function with a phase difference of 90 • was used to generate the rotating magnetic field whose field vector varied regularly around the X axis in the Y-Z plane. Only one electromagnetic coil was activated to produce the magnetic field gradient at X axis. A glass slide was placed in the center of electromagnetic coils. A total of 20 µL of suspension consisting of Ca-Alg/Fe 3 O 4 micromotors and water was added on the glass slide. Upon application of the external magnetic field, the movement of Ca-Alg/Fe 3 O 4 micromotors was observed by an optical microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany) coupled with a CCD camera. The movement video was recorded and analyzed to calculate the advancing velocity of Ca-Alg/Fe 3 O 4 micromotors.

Na-Alg/Fe 3 O 4 Microparticles
Droplet-based microfluidic experiments were performed with three different initial concentrations of Na-Alginate in dispersed fluid (C i,Na-Alg ): 0.09, 0.9 and 9 mg/mL. The concentration of Fe 3 O 4 (C i,Fe 3 O 4 ) was fixed at 0.36 mg/mL for all experiments.

Diameter and Volume
Monodisperse (quasi-)spherical Na-Alg/Fe 3 O 4 microparticles were observed in DMC with an optical microscope ( Figure 2(a1,c1)). The diameter of Na-Alg/Fe 3 O 4 microparticles in DMC (d particle,DMC ) increases with C i,Na-Alg (Table 1). d particle,DMC varies from 31.9 to 102.7 µm with C i,Na-Alg ranging from 0.09 to 9 mg/mL. Table 1. Diameter of Na-Alg/Fe 3 O 4 microparticles in DMC (d particle,DMC ) in function of the initial concentration of Na-Alginate in dispersed fluid (C i,Na-Alg ). C i,Na-Alg (mg/mL) d particle,DMC (µm) 0.09 31.9 ± 1.4 0.9 48.2 ± 1. 6 9 102.7 ± 1.7 The volume of Na-Alg/Fe 3 O 4 microparticles in DMC (V particle,DMC ) is calculated according to the Equation (1). (1) V particle,DMC increases with C i,Na-Alg (black symbols in Figure 2d), whose relationship is well fitted by a linear equation (written in black in Figure 2d). On the other hand, the diffusion of Na-Alginate and Fe 3 O 4 from droplets to DMC is negligeable during the water diffusion [26]. Thus, the volume of Na-Alginate (V Na-Alg ) and the volume of Fe 3 O 4 (V Fe 3 O 4 ) are the same as the corresponding volume in the initially generated droplet. V Na-Alg is calculated by dividing the mass of Na-Alginate (m Na-Alg ) with the density of Na-Alginate (ρ Na-Alg ). m Na-Alg is calculated by multiplying C i,Na-Alg with the initial droplet volume (V i,droplet ) (Equation (2)). V Fe 3 O 4 is calculated the same way (Equation (3)). Finally, the solid volume in the Na-Alg/Fe 3 O 4 microparticle (V solid ), sum of V Na-Alg and V Fe 3 O 4 is calculated with the Equation (4).
V solid increases with C i,Na-Alg (orange symbols in Figure 2d) with a leaner fitting equation (written in orange in Figure 2d). In fact, the linear relationship can be explained mathematically. In Equation (4), ρ Na-Alg , ρ Fe3O4 and C i,Fe3O4 are constant. As for V i,droplet , since it varies little from 0.3 to 0.4 µL for all experiments, it can also be considered as constant. C i,Na-Alg is the only variable. Consequently, Equation (4) gives a linear relationship for the function V solid = f C i,Na-Alg .

Interior Liquid
It can be seen from Figure 2d that there is an evident gap between V particle,DMC and V solid , indicating that the Na-Alg/Fe 3 O 4 microparticle is not composed of condensed solid materials. In fact, flattened broken microparticles have been observed by SEM ( Figure S2), which confirms the porous interior structure. For Na-Alg/Fe 3 O 4 microparticles collected in DMC, there can be water trapped inside after water diffusion reaches equilibrium. DMC can also diffuse into Na-Alg/Fe 3 O 4 microparticles. Thus, the difference between V particle,DMC and V solid is caused by the interior liquid which is water and DMC. The volume of the interior liquid (V interior liquid ) is simply calculated with Equation (5) and plotted with C i,Na-Alg in Figure 2d (red symbols). V interior liquid shows a linear relationship with C i,Na-Alg (fitting equation written in red in Figure 2d). The liquid portion, defined as the liquid volume percentage in the Na-Alg/Fe 3 O 4 microparticle (V interior liquid %), is calculated by Nanomaterials 2022, 12, 115 6 of 13 Equation (6). It is found that about 50-70% of the Na-Alg/Fe 3 O 4 microparticle is filled with liquid, despite C i,Na-Alg used (Figure 2e). (a1,b1,c1) Optical microscopic images and (a2,b2,c2) SEM images of Na-Alg/Fe3O4 microparticles. The initial concentration of Na-Alginate in dispersed fluid ( , ) for the preparation is (a1,a2) 0.09 mg/mL, (b1,b2) 0.9 mg/mL, (c1,c2) 9 mg/mL. The red arrow in b1 shows the neat cross section. SEM scale bar 20 µm. Relationship between , and (d) Na-Alg/Fe3O4 microparticle volume, interior liquid volume, solid volume; (e) liquid portion in Na-Alg/Fe3O4 microparticles in DMC. (f) Evolution of the diameter of Na-Alg/Fe3O4 microparticles (prepared with , at 9 mg/mL) in different media.

Stability
Na-Alg/Fe3O4 microparticles were stored in DMC and air (25 °C, humidity 40%), respectively, to assess the stability in terms of diameter in 72 h after the preparation. Na-Alg/Fe3O4 microparticles were observed by both optical microscope and SEM. The diameter of Na-Alg/Fe3O4 microparticles is measured in three different ways. (1) In DMC with optical microscopic observation: the diameter is stable around 100 µm (black symbols in Figure 2f). (2) In air with optical microscopic observation: the diameter reduces from 100 to 82 µm within the first 24 h and stays unchanged for the following 48 h (orange symbols in Figures 2f and S3a1-5). It can be explained by the evaporation of the interior liquid in air. After being dried in air for 72 h, the same sample is also observed by SEM ( Figure S4). The diameter measured from SEM observation (green symbol in Figure 2f) shows no evident difference from that from optical microscopic observation, indicating that the observing tool does not contribute to the difference of measurement. (3) Samples are coated by a The initial concentration of Na-Alginate in dispersed fluid (C i,Na-Alg ) for the preparation is (a1,a2) 0.09 mg/mL, (b1,b2) 0.9 mg/mL, (c1,c2) 9 mg/mL. The red arrow in b1 shows the neat cross section. SEM scale bar 20 µm. Relationship between C i,Na-Alg and (d) Na-Alg/Fe 3 O 4 microparticle volume, interior liquid volume, solid volume; (e) liquid portion in Na-Alg/Fe 3 O 4 microparticles in DMC. (f) Evolution of the diameter of Na-Alg/Fe 3 O 4 microparticles (prepared with C i,Na-Alg at 9 mg/mL) in different media.

Stability
Na-Alg/Fe 3 O 4 microparticles were stored in DMC and air (25 • C, humidity 40%), respectively, to assess the stability in terms of diameter in 72 h after the preparation. Na-Alg/Fe 3 O 4 microparticles were observed by both optical microscope and SEM. The diameter of Na-Alg/Fe 3 O 4 microparticles is measured in three different ways. (1) In DMC with optical microscopic observation: the diameter is stable around 100 µm (black symbols in Figure 2f). (2) In air with optical microscopic observation: the diameter reduces from 100 to 82 µm within the first 24 h and stays unchanged for the following 48 h (orange symbols in Figures 2f and S3(a1-a5)). It can be explained by the evaporation of the interior liquid in air. After being dried in air for 72 h, the same sample is also observed by SEM ( Figure S4). The diameter measured from SEM observation (green symbol in Figure 2f) shows no evident difference from that from optical microscopic observation, indicating that the observing tool does not contribute to the difference of measurement. (3) Samples are coated by a thin layer of Au once and observed by SEM. They are stored in air each time after observation. The diameter reduces from 100 to 89 µm within the first 24 h and stays unchanged for the following 48 h (blue symbols in Figures 2f and S3(a1-a5)). The final diameter is higher than that observed in air. It is due to the presence of deposited Au layer on the surface of Na-Alg/Fe 3 O 4 microparticles which hinders the evaporation of the interior liquid. Consequently, larger microparticles are observed.
Note that with the same C i,Na-Alg at 9 mg/mL, V solid ( Figure 2d) gives an equivalent diameter of 70 ± 2 µm. It is still lower than the last diameter measured in air with optical microscope (82 µm). Thus, we assume that the interior liquid is not all evaporated.

Morphology
The morphology of Na-Alg/Fe 3 O 4 microparticles was characterized by SEM. Different morphologies were observed depending on C i,Na-Alg used for the preparation. When C i,Na-Alg is extremely low at 0.09 mg/mL, the surface of Na-Alg/Fe 3 O 4 microparticles is rough ( Figure 2(a2)). By increasing C i,Na-Alg to 0.9 mg/mL, the obtained Na-Alg/Fe 3 O 4 microparticles have a smooth and porous surface with a neat cross section (Figures 2(c2) and S5(a1-a3)). The fact that this structure is also observed by optical microscope for Na-Alg/Fe 3 O 4 microparticles in DMC (red arrow in Figure 2(b1)), demonstrating that it is not caused by vacuum during SEM observation. For the highest C i,Na-Alg at 9 mg/mL, Na-Alg/Fe 3 O 4 microparticles have a smooth and porous surface with a tiny flat round trace (Figures 2(c2) and S5(b1-b3)).
Since C i,Fe 3 O 4 and V i,droplet are constant parameters for all experiments, the quantity of Fe 3 O 4 is fixed in Na-Alg/Fe 3 O 4 microparticles. Thus, the difference of morphology is caused by the quantity of Na-Alginate. Based on the results, the mechanism of the formation of Na-Alg/Fe 3 O 4 microparticles is proposed as follows.

Mechanism of the Formation of Na-Alg/Fe 3 O 4 Microparticles
Na-Alg/Fe 3 O 4 droplets are collected in DMC in a glass Petri dish. As droplets fall in DMC, water diffuses from droplets into DMC though the interface (Figure 3a). The diffusion of water brings about the migration of alginate polymeric chains until the interface [27]. Progressively, a primary porous shell structure is formed. When a droplet lands on the glass bottom, the deformation depends on the resistance of the shell which is influenced by the alginate quantity (Q alginate ) in the droplet ( Figure 3b). When Q alginate is extremely low (corresponding to C i,Na-Alg = 0.09 mg/mL), the shell is not resistant enough to the shock upon landing, producing a spreading form. Since there is no sufficient alginate to form a full shell, the final structure is rather a random stock of Fe 3 O 4 nanoparticles which is the major solid material in this case (Figure 3c). When Q alginate is increased but still low (corresponding to C i,Na-Alg = 0.9 mg/mL), the shell resists better the shock of glass. The droplet stays spherical with a neat cross section due to the contact with the glass, which explains the morphology observed by SEM. With the highest Q alginate (corresponding to C i,Na-Alg = 9 mg/mL), the shell is so strong that the droplet is hardly deformed. In the end, spherical microparticles are obtained with a tiny flat round trace left when touching the glass.
(2) For Na-Alg/Fe3O4 microparticles prepared with , at 0.9 and 9 mg/mL These Na-Alg/Fe3O4 microparticles have a similar manner of deformation depending on . With a concentrated solution of CaCl2 ( = 10 wt%), no deformation is produced. Ca-Alg/Fe3O4 micromotors are spherical. When Na-Alg/Fe3O4 microparticles are immerged in a solution with at 1 wt%, a tail is grown from the "defect" (neat cross section or tiny flat round trace, Figure 2b2,c2) on the surface of Na-Alg/Fe3O4 microparticles (Video S1). As a result, Ca-Alg/Fe3O4 micromotors have a droplet-like shape. By reducing to 0.1 wt%, a longer tail is grown, creating the worm-like shape. Finally, the use of a solution with at 0.01 wt% almost dissolves Na-Alg/Fe3O4 microparticles.

Influence of C CaCl 2 on the Shape of Ca-Alg/Fe 3 O 4 Micromotors
(1) For Na-Alg/Fe 3 O 4 microparticles prepared with C i,Na-Alg at 0.09 mg/mL Q alginate is low in Na-Alg/Fe 3 O 4 microparticles. The gelation does not produce any evident deformation with C CaCl 2 varying from 0.1 to 10 wt%. Ca-Alg/Fe 3 O 4 micromotors stay quasi-spherical. When the solution of CaCl 2 is too diluted (C CaCl 2 = 0.01 wt%), Na-Alg/Fe 3 O 4 microparticles are almost dissolved.
(2) For Na-Alg/Fe 3 O 4 microparticles prepared with C i,Na-Alg at 0.9 and 9 mg/mL These Na-Alg/Fe 3 O 4 microparticles have a similar manner of deformation depending on C CaCl 2 . With a concentrated solution of CaCl 2 (C CaCl 2 = 10 wt%), no deformation is produced. Ca-Alg/Fe 3 O 4 micromotors are spherical. When Na-Alg/Fe 3 O 4 microparticles are immerged in a solution with C CaCl 2 at 1 wt%, a tail is grown from the "defect" (neat cross section or tiny flat round trace, Figure 2(b2,c2)) on the surface of Na-Alg/Fe 3 O 4 microparticles (Video S1). As a result, Ca-Alg/Fe 3 O 4 micromotors have a droplet-like shape. By reducing C CaCl 2 to 0.1 wt%, a longer tail is grown, creating the worm-like shape. Finally, the use of a solution with C CaCl 2 at 0.01 wt% almost dissolves Na-Alg/Fe 3 O 4 microparticles.  When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01  When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01  When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01 Table 2. Optical microscopic and schematic images of Ca-Alg/Fe3O4 micromotors with the indication of , − and 2 for the preparation. Scale bar 100 µm.

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

wt%
Nanomaterials 2022, 12, x FOR PEER REVIEW 9 of 13 Table 2. Optical microscopic and schematic images of Ca-Alg/Fe3O4 micromotors with the indication of , − and 2 for the preparation. Scale bar 100 µm.

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01 Table 2. Optical microscopic and schematic images of Ca-Alg/Fe3O4 micromotors with the indication of , − and 2 for the preparation. Scale bar 100 µm.

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01 Table 2. Optical microscopic and schematic images of Ca-Alg/Fe3O4 micromotors with the indication of , − and 2 for the preparation. Scale bar 100 µm.

Mechanism of the Deformation
When adding an aqueous solution of CaCl2 for the gelation, calcium cations diffuse into Na-Alg/Fe3O4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe3O4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe3O4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2b2,c2). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation. When 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe3O4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe3O4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe3O4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when 2 is too low at 0.01

Mechanism of the Deformation
When adding an aqueous solution of CaCl 2 for the gelation, calcium cations diffuse into Na-Alg/Fe 3 O 4 microparticles, causing ionic crosslinking. It allows strengthening bonds between alginate polymeric chains, which impedes further deformation. Meanwhile, water also diffuses into Na-Alg/Fe 3 O 4 microparticles, causing swelling. The swelling is anisotropic because of the anisotropic morphology of Na-Alg/Fe 3 O 4 microparticles with "defect" on the surface (neat cross section or tiny flat round trace, Figure 2(b2,c2)). Overall, in our case, the gelation process is a competition of crosslinking which impedes the deformation and anisotropic swelling which encourages the deformation.
When C CaCl 2 is high at 10 wt%, calcium cations diffuse fast to Na-Alg/Fe 3 O 4 microparticles to achieve crosslinking. The spherical shape is preserved with nearly no swelling. When C CaCl 2 is 1 wt%, calcium cations diffuse more slowly to Na-Alg/Fe 3 O 4 microparticles, leading to a slower crosslinking. It leaves time for anisotropic swelling. According to the result, the swelling is more important at the "defect" on the surface, which explains the growth of the tail. A further reduction in C CaCl 2 to 0.1 wt% is more advantageous for anisotropic swelling, as there is more water and fewer calcium cations in the environment. A longer tail is grown. Furthermore, we note that with the anisotropic swelling, Na-Alg/Fe 3 O 4 microparticle grow upwards from the glass bottom (Video S1) with a tail adhered to the glass. The tail bends when it can no longer bear the weight of the whole microparticle. It explains why certain tails are curved. Finally, when C CaCl 2 is too low at 0.01 wt%, there are no sufficient calcium cations for crosslinking. Anisotropic swelling takes over crosslinking totally. Na-Alg/Fe 3 O 4 microparticles are almost dissolved.

Characterization of Fe 3 O 4 Nanoparticles inside Ca-Alg/Fe 3 O 4 Micromotors
By using FITC-labeled Fe 3 O 4 nanoparticles, the fabricated micromotors were observed by CLSM. The fluorescent image (Figure 4(a1,a2)) indicates that Fe 3 O 4 nanoparticles are incorporated in Ca-Alginate hydrogel successfully. It is also verified by EDX mapping analysis (Figure 4(b1,b2)). Moreover, the 3D-image reconstruction by CLSM shows that Fe 3 O 4 nanoparticles are mostly located at the bottom half semi-sphere of the micromotor (Video S2). Fe3O4 nanoparticles are mostly located at the bottom half semi-sphere of the micromotor (Video S2).

Locomotion of Ca-Alg/Fe3O4 Micromotors
The locomotion of all three types of micromotors is always observed at the bottom of the glass Petri dish. For spherical and worm-like Ca-Alg/Fe3O4 micromotors, the locomotion is activated by applying a rotating magnetic field at 5 mT around X axis in Y-Z plane. The frequency ( ) varies from 1 to 15 Hz. We observe that spherical micromotors move forward by rotation-enabled rolling (Figure 4d, Video S3). The advancing velocity ( ) increases with until the step-out frequency ( = 6 Hz) is reached (Figure 4c). Then, declines with the frequency. The maximum is

Locomotion of Ca-Alg/Fe 3 O 4 Micromotors
The locomotion of all three types of micromotors is always observed at the bottom of the glass Petri dish. For spherical and worm-like Ca-Alg/Fe 3 O 4 micromotors, the locomotion is activated by applying a rotating magnetic field at 5 mT around X axis in Y-Z plane.
The frequency ( f ) varies from 1 to 15 Hz. We observe that spherical micromotors move forward by rotation-enabled rolling (Figure 4d, Video S3). The advancing velocity (v advancing ) increases with f until the step-out frequency ( f step-out = 6 Hz) is reached (Figure 4c). Then, v advancing declines with the frequency. The maximum v advancing is 158.2 ± 8.6 µm/s with f at 6 Hz. However, as for worm-like micromotors, with the application of rotating magnetic field, rotation behavior is observed, which can be further developed for advancing movement based on their helical structure (Figure 4e, Video S4).
With the symmetric structure, droplet-like Ca-Alg/Fe 3 O 4 micromotors cannot move forward under the rotating magnetic field. The locomotion of a droplet-like Ca-Alg/Fe 3 O 4 micromotor is activated by applying an X-axis magnetic field gradient. At 2 T/m, the micromotor is not actuated at all. At 3 T/m, a torque is first produced and turns the micromotor to align with the direction of the external magnetic field. Then, the micromotor is pulled forward (Figure 4f, Video S5) with an average velocity of 70.7 ± 2.8 µm/s. Although all micromotors are not actuated in the same type of external magnetic field, we note that with the same current input in the electromagnetic coil, micromotors are more likely to be actuated by the rotating magnetic field than the magnetic field gradient. It can be explained by the fact that the friction force is generally higher when a micromotor is pulled forward than when it advances by rotation. Thus, the rotating magnetic field is preferably reported in the literature, whereas it demands a certain shape of micromotors, such as spherical and helical shapes. The present method allows producing the spherical shape. We hope that with further modification and precise control during gelation, the worm-like shape can be transformed into the well-defined shape such as helical structured motors.

Conclusions and Perspective
In conclusion, we present herein a facile droplet-based microfluidic preparation of Fe 3 O 4 -incorporated alginate hydrogel micromotors with variable shapes. First, monodisperse (quasi-)spherical Na-Alg/Fe 3 O 4 microparticles are obtained by using droplet-based microfluidics and water diffusion. The diameter of Na-Alg/Fe 3 O 4 microparticles varies from 31.9 to 102.7 µm, depending on the initial concentration of Na-Alginate in dispersed fluid. The mechanism of the formation of Na-Alg/Fe 3 O 4 microparticles is proposed to explain different morphology observed by SEM. Second, an aqueous solution of CaCl 2 is used for gelation, transforming Na-Alg/Fe 3 O 4 microparticles into Ca-Alg/Fe 3 O 4 micromotors. Spherical, droplet-like, and worm-like shapes are obtained, which is mainly affected by the concentration of CaCl 2 . The mechanism of deformation is proposed considering the crosslinking and anisotropic swelling during the gelation. Finally, Ca-Alg/Fe 3 O 4 micromotors are actuated by applying external magnetic fields, showing their potential of developing functional micro/nano-motors.
This article provides an idea for producing different shapes of hydrogel micromotors without using complex patterns or sophisticated facilities. The method is timesaving and easy to be realized. In terms of the stability of micromotors, the size has been measured for 14 days consecutively and no evident change was found. Stored in pure water, micromotors demonstrated a decreasing locomotion probably due to the diffusion of Fe 3 O 4 nanoparticles. Nevertheless, the advancing velocity of spherical micromotors was estimated to be stable within 2 days. For long-term utility or storage, work should be done to conserve the locomotion of micromotors. Future work will focus on integrating biomedical or biological agents, such as medical molecules and cells to achieve on-demand tasks such as drug delivery and cell transportation.